Estimated timetree of vertebrates.

<p>Timetree produced by MCMCTREE in PAML 4.4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone.0066400-Yang1" target="_blank">[62]</a> implementing the relaxed molecular clock method. A total of 19 time constraints (see Table S2) used for the calculation are shown as arrowheads at the eleven nodes. 2973 amino acid sites were analyzed derived from a total of 20 nuclear genes. Horizontal bars indicate 95% confidence intervals (CI) of the divergence time estimates. All estimates and 95% CIs are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone-0066400-t001" target="_blank">Table 1</a>. The marginal densities obtained in TRACER 1.5 are shown in light grey above the bars. Rates given by MCMCTREE are shown above the individual branches.</p

Phylogenetic tree of<i>RAN binding protein 1</i> genes.

<p>This gene is listed as candidate #9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone-0066400-t001" target="_blank">Table 1</a>. The tree was reconstructed with the maximum-likelihood (ML) method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#s4" target="_blank">Methods</a>). Bootstrap values were calculated with 100 resamplings. Support values at nodes indicate, in order, probabilities in the ML and the neighbor-joining (NJ) analysis. 119 amino acid sites were included for tree inference (shape parameter for gamma distribution α = 0.38). Note that the topology of this ML tree is not consistent with the generally accepted species phylogeny, but the log-likelihood of the tree topology consistent with the species phylogeny was not significantly lower than that of the ML tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone-0066400-t001" target="_blank">Table 1</a>). For this reason, this gene was included in the final dataset.</p

Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes

<div><p>Cartilaginous fishes, divided into Holocephali (chimaeras) and Elasmoblanchii (sharks, rays and skates), occupy a key phylogenetic position among extant vertebrates in reconstructing their evolutionary processes. Their accurate evolutionary time scale is indispensable for better understanding of the relationship between phenotypic and molecular evolution of cartilaginous fishes. However, our current knowledge on the time scale of cartilaginous fish evolution largely relies on estimates using mitochondrial DNA sequences. In this study, making the best use of the still partial, but large-scale sequencing data of cartilaginous fish species, we estimate the divergence times between the major cartilaginous fish lineages employing nuclear genes. By rigorous orthology assessment based on available genomic and transcriptomic sequence resources for cartilaginous fishes, we selected 20 protein-coding genes in the nuclear genome, spanning 2973 amino acid residues. Our analysis based on the Bayesian inference resulted in the mean divergence time of 421 Ma, the late Silurian, for the Holocephali-Elasmobranchii split, and 306 Ma, the late Carboniferous, for the split between sharks and rays/skates. By applying these results and other documented divergence times, we measured the relative evolutionary rate of the Hox A cluster sequences in the cartilaginous fish lineages, which resulted in a lower substitution rate with a factor of at least 2.4 in comparison to tetrapod lineages. The obtained time scale enables mapping phenotypic and molecular changes in a quantitative framework. It is of great interest to corroborate the less derived nature of cartilaginous fish at the molecular level as a genome-wide phenomenon.</p></div

Estimated divergence times.

<p>Divergence time estimates (posterior mean) and their 95% confidence intervals (CIs) in Ma (million years from present) for eleven nodes indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone-0066400-g004" target="_blank">Figure 4</a>. See Table S2 for details of the time constraints. Different minimum constraints of node #11 resulted in different divergence time estimates.</p

Relationship of chondrichthyan species.

<p>Species tree illustrating the relationship of all chondrichthyan species employed in our analyses, either in the divergence time study or evolutionary rate analysis (see text for alternative views of the phylogenetic relationship). Circles indicate the nodes referred to in the divergence time analysis. Widths of triangles are proportional to the numbers of species for individual groups according to Compagno et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066400#pone.0066400-Compagno1" target="_blank">[69]</a>.</p

Distances and evolutionary rates for chondrichthyan and tetrapod representatives.

<p>Distances (number of substitutions per site) were calculated by codeml for different pairs of species using <i>Callorhinchus milii</i> (<i>Cm</i>) as outgroup. Applying divergence times estimated in this study (306 Ma) as well as previous studies (203 Ma, 312 Ma and 330 Ma), evolutionary rates were calculated for three chondrichthyans and four tetrapod species. Abbreviations: aa, amino acid; Ma, million years ago; <i>Hf</i>, <i>Heterodontus francisci</i>; <i>Sc</i>, <i>Scyliorhinus canicula</i>; <i>Le</i>, <i>Leucoraja erinacea</i>; <i>Gg</i>, <i>Gallus gallus</i>; <i>Hs</i>, <i>Homo sapiens</i>; <i>Ac</i>, <i>Anolis carolinensis</i>; <i>Xt</i>, <i>Xenopus tropicalis</i>; O, Last common ancestor of the two selected species.</p

Overview of nuclear genes used for the divergence time analysis.

<p>The log-likelihood difference (Δlog<i>L</i>) between the ML tree and the topology based on the generally accepted species phylogeny is shown with standard error. The topology of the ML tree is shown in newick format. The number of amino acid sites used for tree inference is shown for each gene, as well as their corresponding shape parameter α for gamma distribution. Abbreviations: aa, amino acid; ML, maximum likelihood tree; Dm, <i>Drosophila melanogaster</i>; Ds, <i>Drosophila simulans</i>; Ci, <i>Ciona intestinalis</i>; Cm, chimaeras; Sh, sharks; Ry, rays/skates; Fr, <i>Takifugu rubripes</i>; Ol, <i>Oryzias latipes</i>; Dr, <i>Danio rerio</i>; Hs, <i>Homo sapiens</i>; Md, <i>Monodelphis domestica</i>; Gg, <i>Gallus gallus</i>; Xt, <i>Xenopus tropicalis</i>.</p

Split-Doa10: A Naturally Split Polytopic Eukaryotic Membrane Protein Generated by Fission of a Nuclear Gene

<div><p>Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast <em>Kluyveromyces lactis</em>. In most species, Doa10 is encoded as a single polypeptide with 12–16 transmembrane helices (TMs), but split-<em>Kl</em>Doa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The <em>K. lactis DOA10</em> locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal <em>Kl</em>Doa10 fragment. At least four additional <em>Kluyveromyces</em> species contain an IVS in the <em>DOA10</em> locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream <em>Kluyveromyces</em> Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.</p> </div

Transcriptome data of the zebra bullhead shark

Supplementary data of Onimaru et al., "A de novo transcriptome assembly of the zebra bullhead shark,<i> </i><i>Heterodontus zebra</i>

Possible gene fission scenarios for <i>Kluyveromyces DOA10.</i>

<p>Fission of the Doa10 ORF might have occurred by either of the two following ways: (1) Splitting by insertion of a foreign sequence into the <i>DOA10</i> gene by a recombination event. The Doa10 ORF is disrupted by integration of the foreign sequence (light grey box) whereby a stop codon is created leading to the Nt-ORF and the Ct-ORF. The Ct-ORF starts close to the 3′ end of the inserted sequence (as depicted) or, alternatively, within the inserted sequence (not depicted). The foreign sequence might have already had promoter activity (indicated by “(P)”) before insertion or might have acquired it later. (2) Splitting by internal diversification of the Doa10 coding sequence. The acquisition of a stop codon, e.g. by point mutation, resulted in generation of the Doa10 Nt-ORF. The region downstream of the newly created stop codon (shaded in grey) might have displayed (residual) promoter activity or alternatively, might have subsequently evolved into a promoter driving expression of the downstream Ct-ORF. See main text for details. P, promoter; T, terminator; the beginning of an ORF is depicted with an arrow, its end with an asterisk.</p